CPK1 | Calcium-dependent protein kinase 1

Plant age: 4 weeks under short-day conditions (8 h light).
20 µg of the total protein from Arabidopsis thaliana plants,  (genetic background: Col-0) plants, wild-type (1), cpk1 (SALK_096452) (2), cpk1 (SALK_080155c) (3) were extracted with protein extraction buffer (Durian et al., 2019) and denatured in Laemmli-sample buffer (final concentrations: 5% (v/v) beta-mercaptoethanol, 69 mM Tris-HCl pH 6.8, 11.1% (v/v) glycerol, 2.15 % (v/v) SDS) for 10 min at 74 ºC. Samples were separated by discontinuous gel electrophoresis on a 10 % (w/v) separation SDS-PA-gel and blotted 1h to PVDF-membrane using a semi-dry system. The blot was blocked with 5% milk powder in TBS-T with agitation for 1h at room temperature (RT). After rinsing in TBS-T, the blot was incubated in the primary antibody at a dilution of 1:1000 in TBS-T with 1% milk powder in 7°C with agitation overnight. The antibody solution was decanted and the blot was rinsed briefly twice with TBS-T, then washed three times for 10 min in TBS-T at RT with agitation. The blot was incubated with secondary antibody (Anti-rabbit IgG horse radish peroxidase) diluted to 1:10 000 for 1h at RT in TBS-T with 1% milk-powder with agitation. The blot was washed as above plus once for 10 min with TBS and developed for 5 min with chemiluminescent detection reagent, according to manufacture's recommendations. Visualization by exposure to Super RX film (Fujifilm 47410-19236). Exposure time of the film: 30 min.

Courtesy of Dr. Guido Durian, University of Turku, Finland